Project Summary Helicases and nucleases play important roles in maintaining genome integrity by resolving intermediates produced during DNA metabolic processes, such as DNA replication and repair. Recent studies suggest that genomic ribonucleotide represents one of the most abundant DNA lesions in cells. In addition to the ribonucleotides that prime DNA replication, significant amount of ribonucleotides are mis-incorporated into the genome per cell cycle by replicating DNA polymerases. In this proposal, using budding yeast as a model system, we outlined research to define the roles of Srs2 helicase and its related nucleases in the error-free removal of both priming and mis-incorporated genomic ribonucleotides. Specifically, we will combine genetic, biochemical, and structural approaches to define the mechanisms of Top1-catalyzed genomic ribonucloetide excision repair with a focus on the Srs2-initiated error-free branch. In addition, the potential role of Srs2 in lagging strand maturation will also be delineated. Knowing that mutation of genes involved in ribonucloetide excision repair is associated with Aicardi-Goutleres Syndrome and genome instability caused by genomic ribonucloetide incorporation may increase the risk of cancer, our research has direct disease relevance. In compliance with goal of MIRA award, we will continue to tackle other key questions related to genome maintenance as we have done in the past.